The overall objective of Project 1 is to understand the mechanism by which toxic metals effect cellular oxidative stress. Oxidative stress has been implicated in a number of human diseases, including cancer, aging, atherosclerosis, and fibrosis. There is some evidence that toxic metals can induce oxidative stress, which may be the mechanism for both genotoxic and non-genotoxic proliferative disease. The ability of the toxic and/or carcinogenic metals, chromium, nickel, cadmium, lead, iron and arsenic, individually and in combinations found at contaminated sites, to induce oxidative stress will be determined in chick embryo in vivo and cultured endothelial cells. The specific hypothesis for these studies is that metals may cause cancer or other proliferative diseases by altering expression of specific genes at the level of transcription. This alteration may occur either from a direct effect on DNA structure and consequently the DNA recognition sequences for trans-acting factors or through activation of cell signaling for a greater abundance and/or activity of transcription factors. We plan to not only examine possible induction of oxidative DNA damage by toxic metals, but also the propensity of these metals to cause aberrant gene induction through alteration of cell signaling. The specific aims of the proposed research are to: (1) Determine the ability of toxic metals to produce reactive oxygen intermediates (ROIs) in cultured cells using biochemical analysis for accumulation of reactive species and fluorescence spectroscopy to demonstrate intracellular oxidant levels. Induction of reactive oxygen species, such as hydrogen peroxide and superoxide, by toxic metals can be assayed by spectrophotometric and fluorometric techniques. The level of intracellular oxidant levels can be measured by use of metabolizable reagents such as rhodamine and dichlorofluoresin diacetate which enter cells irreversibly and are converted to fluorescent species by reactive oxygen species. (2) Investigate the induction of oxidative DNA damage by toxic metals in chick embryo tissues in vivo and in cultured normal pig aorta endothelial cells by measuring DNA single strand breaks, DNA- protein cross-links and levels of 8-oxo-2'-deoxyguanosine. If reactive oxygen species are produced intracellularly by toxic metals, then the ROIs may attack DNA causing oxidative DNA lesions. DNA strand breakage will be analyzed by using a DNA unwinding assay, DNA-protein cross-links by using a K+-SDS precipitation assay and 8-oxo-2'-deoxyguanosine by using high performance liquid chromatography with electrochemical detection (HPLC-ECD). (3) Demonstrate the levels of metals that cause specific, phosphorylation-dependent activation of transcription factors and establish the rate determining sites in this signaling cascade. Activation of trans-acting factors, which induce genes known to be expressed following oxidant-stress, i.e., heme oxygenase, and urokinase- like plasminogen activator (uPA), will be assayed by electrophoretic mobility assays and the sensitivity to kinase inhibition will be determined. The effect of inhibiting transcription factor activation on steady state mRNA levels of oxidant-sensitive genes will be assayed in chick embryo tissues in vivo and cultured normal pig aorta endothelial cells. The proposed studies should provide evidence for oxidative pathways for toxic metal action on expression of specific genes, and provide insight into the mechanism by which metals cause their toxic effects. This, in turn, could provide fundamental insights into strategies designed to prevent metal induction of oxidative stress.